Intracellular Localization Research Articles

A Method for Bioluminescence-Based RNA Monitoring Using Split-Luciferase Reconstitution Techniques.

A bioluminescence-based RNA monitoring method in living cells was developed using a split NanoLuc (NLuc) reconstitution technique. For specific recognition of a target RNA sequence, a mutant PUM-HD (mPUM) was used. The method was applied to β-actin mRNA in various cells, including primary cultures of rat hippocampal neurons. It allowed for continuous observation of intracellular localization distribution of the target mRNA in living cells, providing insights into various biological phenomena involving intracellular RNA localization and dynamics.

Functional characterization of OR51B5 and OR1G1 in human lung epithelial cells as potential drug targets for non-type 2 lung diseases

Abstract Background Hypersensitivity to odorants like perfumes can induce or promote asthma with non-type 2 inflammation for which therapeutic options are limited. Cell death of primary bronchial epithelial cells (PBECs) and the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 are key in the pathogenesis. Extra-nasal olfactory receptors (ORs) can influence cellular processes involved in asthma. This study investigated the utility of ORs in epithelial cells as potential drug targets in this context. Methods We used the A549 cell line and primary bronchial epithelial cells using air–liquid interface culture system (ALI-PBECs). OR expression was investigated by RT-PCR, Western blot, and Immunofluorescence. Effects of OR activation by specific ligands on intracellular calcium concentration, cAMP, Phospholipase C (PLC), cell viability, and IL-6 and IL-8 secretion were analyzed by calcium imaging, enzyme immunoassays, Annexin V/ propidium iodide -based fluorescence-activated cell staining or by ELISA, respectively. Results By screening A549 cells, the OR51B5 agonists Farnesol and Isononyl Alcohol and the OR1G1 agonist Nonanal increased intracellular Ca2 + . OR51B5 and OR1G1 mRNAs and proteins were detected. Both receptors showed a preferential intracellular localization. OR51B5- but not OR1G1-induced Ca2 + dependent on both cAMP and PLC signaling. Farnesol, Isononyl Alcohol, and Nonanal, all reduced cell viability and induced IL-8 and IL-6 release. The data were verified in ALI-PBECs. Conclusion ORs in the lung epithelium might be involved in airway-sensitivity to odorants. Their antagonism could represent a promising strategy in treatment of odorant-induced asthma with non-type 2 inflammation. Graphical Abstract

STEM-13. NUCLEAR LOCALIZATION OF ALPHA-1-ANTICHYMOTRYPSIN INCREASES IN GLIOBLASTOMA FOLLOWING STANDARD OF CARE TREATMENT

Abstract Glioblastoma (GBM) is the most common malignant primary brain tumor in adults with a median survival of 8-14 months and 5-year survival rate of 6.9%. Its aggressive nature arises mostly from molecular heterogeneity and resistance to standard of care (SOC), including radiation and temozolomide (TMZ). Recurrence is nearly inevitable, driven by highly invasive brain tumor initiating cells (BTICs). Our previous studies revealed that exposing BTICs to cerebrospinal fluid (CSF) from GBM patients increases cell migration and proliferation compared to non-cancer CSF. Transcriptomic analysis identified the upregulation of the SERPINA3 gene, which encodes alpha-1-antichymotrypsin (ACT). Silencing of SERPINA3 reduces BTIC migration and proliferation and abolishes the promigratory response to GBM-derived CSF, highlighting SERPINA3 as a potential therapeutic target. ACT has been reported to bind to DNA, although the implications of this interaction are unknown. We have previously observed higher levels of intranuclear ACT in human GBM compared to non-cancer brain tissues. We hypothesize that the nuclear localization of SERPINA3 in GBM contributes to increased malignancy, opening the possibility of therapeutically targeting its intranuclear transport. Here, we determined the intracellular localization of ACT in BTICs and GBM and compared it to non-cancer tissues. Additionally, we evaluated whether SOC affected intranuclear localization of ACT. Patient-derived BTICs were treated with TMZ, radiation, and a combination of both, to assess changes in ACT expression via Western Blot, PCR, and ICC. A decrease in cell confluency was correlated with a significant decrease (p<0.05) in SERPINA3 mRNA and overall protein expression when BTICs were treated with TMZ alone or in combination. Interestingly, confocal microscopy revealed a significant increase (p<0.05) in its nuclear localization, particularly with TMZ treatment, indicating a change in its intracellular distribution. Overall, nuclear translocation of SERPINA3 increases in response to SOC with potential implications as a treatment resistance mechanism.

Exploring the Intracellular Distribution of Se Compounds Delivered by Biodegradable Polyelectrolyte Capsules Using X‐Ray Fluorescence Imaging

AbstractSelenium (Se) compounds hold promise as potential therapeutics for cancer due to their diverse biological functions and redox‐regulating properties. In this study, the encapsulation of a hydrophobic selenium compound inside 5 µm sized biodegradable polyelectrolyte capsules consisting of poly‐L‐arginine and dextran is investigated. While the encapsulated form of the compound showed comparable cellular uptake and cytotoxicity as the free drug, e.g. no improved anti‐cancer efficacy is achieved, the investigation yielded crucial insights. Utilizing subcellular resolution synchrotron X‐ray fluorescence imaging (XFI), the intracellular location of the Se compound is successfully visualized by demonstrating the degradation of the capsules and the following redistribution of the compound, without exogenous labeling. By applying a quantitative fitting approach, the intracellular Se mass is determined to be 0.09 ± 0.01 pg after degradation of the carrier. This highlights the potential of XFI as a powerful tool for tracking intracellular dynamics of metal‐based drugs, which may facilitate drug development in the clinic.

Optimization of Antiproliferative Properties of Triimine Copper(II) Complexes.

Cu(II) complexes with 2,2':6',2″-terpyridines (terpy) and 2,6-bis(thiazol-2-yl)pyridines (dtpy) with 1- or 2-naphtyl and methoxy-naphtyl were synthesized to elucidate the impact of the triimine core, naphtyl linking mode, and presence of methoxy groups on the antiproliferative activity of [CuCl2(Ln)]. Their antiproliferative effect was analyzed in ovarian (A2780) and colorectal (HCT116) carcinomas and colorectal carcinoma resistant to doxorubicin (HCT116-DoxR) cell lines and in normal human fibroblasts. Among all complexes, the 1- and 2-naphtyl substituted terpy Cu(II) complexes (Cu1a and Cu1b) showed the strongest cytotoxicity, namely, in HCT116-DoxR 2Dcells and were also capable of inducing the loss of cell viability in 3D HCT116-DoxR spheroids. Their intracellular localization, capability to increase reactive oxygen species (ROS), and interaction with DNA (nonintercalative mode) trigger oxidative DNA cleavage leading to cell death by apoptosis and autophagy. Cu1a and Cu1b do not show in vivo toxicity in a chicken embryo and can interact with bovine serum albumin (BSA).

Treatment of Conjunctival Melanoma Cell Lines With a Light-Activated Virus-Like Drug Conjugate Induces Immunogenic Cell Death.

Conjunctival melanoma (CJM) is a rare malignant ocular surface tumor, which often leads to local recurrences and metastases. In murine models of subcutaneous tumors, treatment with a novel virus-like drug conjugate (VDC; Bel-sar) showed a dual mechanism of action with direct tumor cell killing as well as stimulation of an antitumoral immune response. Bel-sar is currently being evaluated for the treatment of primary uveal melanoma and indeterminate nevi in a phase III clinical trial. We determined whether Bel-sar also has direct antitumor efficiency and a potential immunostimulatory capacity in CJM cells. Three human tumor-derived CJM lines were used. Bel-sar's subcellular and intracellular locations were determined with tracers. Following light activation of Bel-sar, cytotoxicity and exposure of damage-associated molecular patterns (DAMPs) were assessed. Treated tumor cells were co-cultured with THP-1 derived macrophages to assess tumor-cell phagocytosis. Bel-sar was bound and internalized by CJM cells and subsequently found in the cell membrane, lysosome, Golgi apparatus, and mitochondria. Bel-sar activation induced near complete cell death with half-maximal inhibitory concentration (IC50) values between 30 pM and 60 pM. Finally, light-activated Bel-sar enhanced exposure of DAMPs, including calreticulin, heat shock protein 90, and stimulated phagocytosis by macrophages. Treatment with a novel VDC (Bel-sar) induced pro-immunogenic cell death in all three CJM cell lines. The in vitro cytotoxicity was accompanied by exposure of DAMPs, suggesting Bel-sar is a potential treatment for CJM by a dual mechanism of action. This dual mechanism may provide a targeted and direct killing of tumor cells and induce an immune response which might decrease local recurrences and metastasis.

Glucocorticoid Receptor Isoforms in Breast Cancer Raise Implications for Personalised Supportive Therapies.

Glucocorticoid receptor (GR) activation may promote metastasis in oestrogen receptor-negative and triple-negative breast cancer (TNBC). However, the role of the GRβ isoform, which has opposing effects to the main isoform, has not been studied in clinical samples. We aimed to analyse the intracellular localisation of total GR and GRβ in vitro using plasmid constructs and fluorescent immunocytochemistry. Additionally, our goal was to perform immunostaining for total GR and GRβ on two cohorts: (i) on 194 clinical breast cancer samples to compare the expression in different molecular subtypes, and (ii) on 161 TNBC samples to analyse the association of GR with survival. We supplemented our analysis with RNA data from 1097 TNBC cases. We found that in the absence of the ligand, GR resided in the cytoplasm of breast cancer cells, while upon ligand activation, it translocated to the nucleus. A negative correlation was found between cytoplasmic GRtotal and Ki67 in luminal A tumours, while the opposite trend was observed in TNBC samples. Tumours with strong lymphoid infiltration showed higher cytoplasmic GRtotal staining compared to those with weaker infiltration. Patients with high nuclear GRtotal staining had shorter progression-free survival in univariate analysis. High cytoplasmic GRβ was a marker for better overall survival in multivariate analysis (10-year overall survival HR [95% CI]: 0.46 [0.22-0.95], p = 0.036). As a conclusions, this study is the first to investigate GRβ expression in breast tumours. Different expression and cellular localisation of GRtotal and GRβ were observed in the context of molecular subtypes, underscoring the complex role of GR in breast cancer. An inverse association between cytoplasmic GRtotal and the Ki67 proliferation index was observed in luminal A and TNBC. Regarding the impact of GR on outcomes in TNBC patients, while cytoplasmic GRβ was associated with a better prognosis, patients with nuclear GRtotal staining may be at a higher risk of disease progression, as it negatively affects survival. Caution should be exercised when using glucocorticoids in patients with nuclear GR staining, as it may negatively impact survival.

High-affinity T cell receptor ImmTAC® bispecific efficiently redirects T cells to kill tumor cells expressing the cancer testis antigen PRAME

Abstract PRAME (Preferentially expressed Antigen in Melanoma) is a cancer testis antigen expressed in several tumor indications, representing an attractive anti-cancer target. However, its intracellular location limits targeting by traditional methods. PRAME peptides are presented on the surface of tumor cells by human leukocyte antigen (HLA) molecules, indicating that a T cell receptor (TCR)-based strategy that redirects T cells to kill PRAME+ tumors could be a novel immunotherapeutic option. We confirm that PRAME protein is expressed in cutaneous melanoma, including rare subtypes with limited treatment options, as well as primary and metastatic lung, breast, endometrial and ovarian tumors. Furthermore, PRAME is expressed homogeneously across tumors with distinct oncogenic mutations, mutation burden, PD-L1 expression, immune infiltration and features of immune checkpoint resistance. Immunopeptidomic analysis of primary tumors detected HLA class I-restricted PRAME peptides. A TCR recognizing PRAME peptide SLLQHLIGL was engineered to high affinity and fused to a CD3 engaging domain to create a TCRxCD3 bispecific molecule (Immune mobilizing monoclonal TCR Against Cancer, ImmTAC®) with the ability to redirect polyclonal T cells to efficiently kill PRAME+ cells. The degree of T cell activation was positively correlated with peptide-HLA abundance, with as few as 10 epitopes per cell sufficient for target cell killing. Impaired ImmTAC®-redirected cytotoxicity of exhausted T cells was rescued using an anti-PD-1 antibody, supporting the use of a combination strategy to treat tumors with active PDL1-PD1 axes. Our data demonstrate selective and efficient T cell activation and killing by a PRAME-directed TCRxCD3 bispecific, supporting further investigation in multiple cancer indications.

RNA granules in flux: dynamics to balance physiology and pathology.

The life cycle of an mRNA is a complex process that is tightly regulated by interactions between the mRNA and RNA-binding proteins, forming molecular machines known as RNA granules. Various types of these membrane-less organelles form inside cells, including neurons, and contributecritically to various physiological processes. RNA granules are constantly in flux, change dynamically and adapt to their local environment, depending on their intracellular localization. The discovery that RNA condensates can form by liquid-liquid phase separation expanded our understanding of how compartments may be generated in the cell. Since then, a plethora of new functions have been proposed for distinct condensates in cells that await their validation in vivo. The finding that dysregulation of RNA granules (for example, stress granules) islikely toaffect neurodevelopmental and neurodegenerative diseases further boosted interest in this topic. RNA granules have various physiological functions in neurons and in the brain that we would like to focus on. We outline examples of state-of-the-art experiments including timelapse microscopy in neurons to unravel the precise functions of various types ofRNA granule. Finally, we distinguish physiologically occurring RNA condensation from aberrant aggregation, induced by artificial RNA overexpression, and present visual examples to discriminate both forms in neurons.

Essential magnetosome proteins MamI and MamL from magnetotactic bacteria interact in mammalian cells

To detect cellular activities deep within the body using magnetic resonance platforms, magnetosomes are the ideal model of genetically-encoded nanoparticles. These membrane-bound iron biominerals produced by magnetotactic bacteria are highly regulated by approximately 30 genes; however, the number of magnetosome genes that are essential and/or constitute the root structure upon which biominerals form is largely undefined. To examine the possibility that key magnetosome genes may interact in a foreign environment, we expressed mamI and mamL as fluorescent fusion proteins in mammalian cells. Localization and potential protein-protein interaction(s) were investigated using confocal microscopy and fluorescence correlation spectroscopy (FCS). Enhanced green fluorescent protein (EGFP)-MamI and the red fluorescent Tomato-MamL displayed distinct intracellular localization, with net-like and punctate fluorescence, respectively. Remarkably, co-expression revealed co-localization of both fluorescent fusion proteins in the same punctate pattern. An interaction between MamI and MamL was confirmed by co-immunoprecipitation. In addition, changes in EGFP-MamI distribution were accompanied by acquisition of intracellular mobility which all Tomato-MamL structures displayed. Analysis of extracts from these cells by FCS was consistent with an interaction between fluorescent fusion proteins, including an increase in particle radius. Co-localization and interaction of MamI and MamL demonstrate that select magnetosome proteins may associate in mammalian cells.

Dysfunctional atrial fibroblast processing of filamin A in atrial fibrillation

Abstract Background/Introduction Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is associated with atrial structural remodelling, hallmarked by fibrosis. Atrial fibrosis is instigated by atrial fibroblasts (AFBs). It was previously uncovered the hormone calcitonin (CT) is produced in the heart and, via binding to the CT-receptors (CTR), inhibits atrial fibrogenesis and fibroblast profibrotic activity. [1] In AF, abnormally increased CTR internalisation causes loss of physiological surface CTR and prevents fibroblast activation by CT. The molecular mechanisms of this phenomenon in ACFs are unknown. Aim To explore the role of filamin A (FLNa), an actin cross-linking protein that was previously shown to interact with CTR, [2] in the regulation of CTR trafficking. Methods AFBs were enzymatically isolated from left atrial appendage biopsies collected from 30 patients in persistent AF or normal sinus rhythm (SR, controls) undergoing elective heart surgery. Immunostaining assessed the localisation of CTR and its overlap with the nucleus (stained with DAPI) and FLNa in AFBs. Co-localisation was quantified by the JACoP BIOP plugin for ImageJ. Target mRNA expression was assessed by RT-qPCR and protein levels by immunoblot. Immunoprecipitation (IP) then immunoblot (IB) validated protein-protein interactions. Calpain activity was assessed with a fluorescence-based assay. The effects of calpains on FLNa proteolytic processing was examined with calpain inhibitor calpeptin (10nM–100nM vs DMSO loading control). Results Immunostaining revealed aberrant, intracellular localisation of CTR in AF-AFBs (A) with increased nuclear localisation (p=0.03) and reduced co-localisation with FLNa (p=0.03). FLNa gene expression showed a trend towards reduction in AF (by 36%, p=0.06; B). Meanwhile, although full-length FLNa protein was unaltered in AF, there was a striking 67% reduction (p=0.02) in the expression of FLNa’s c-terminal fragment (FLNaCT), which contains the CTR binding domain (C). Co-immunoprecipitation confirmed a physical interaction between CTR and FLNaCT (D) in both SR and AF AFBs. FLNaCT is typically generated when FLNa is cleaved by the protease calpain 1, whose protein (F) but not mRNA (E), was significantly downregulated (71%, p<0.001) in AF. Puzzlingly, a ~70% loss of calpain 1 protein was associated with unchanged calpain activity (G) and FLNaCT levels in AFBs treated with calpain inhibitor calpeptin (H). Conclusions Persistent AF is associated with an aberrant CTR localisation and FLNa proteolytic processing, not attributable to altered calpain activity. Further characterisation of the molecular determinants of the altered CTR localisation may uncover novel and potentially druggable facets of structural remodelling in AF.

Secreted GRP78 attenuates endothelial inflammation and predicts outcome in coronary artery disease

Abstract Background Endoplasmic-reticulum-stress (ER-Stress) chaperones like the main ER-Stress moderator GRP78 (glucose-regulated-protein, 78kDa) are involved in the pathogenesis of coronary artery disease (CAD). In addition to their established intracellular localization, chaperones are also secreted into the extracellular space. However, the significance of extracellular ER chaperones in CAD remains unclear. Here, we investigated the role of extracellular GRP78 in patients with suspected CAD and its impact on endothelial cell inflammation. Methods Serum concentration of GRP78 was measured by ELISA in 622 patients undergoing coronary angiography (70.0 ± 12.2 years, 33.6% female and 28.4% with acute coronary syndrome: ACS). The primary endpoint was defined as one-year mortality. Human coronary artery endothelial cells (HCAEC) were treated with ER-Stressors and the effects of ER-Stress conditioned medium (CM) on recipient cell viability, apoptosis, reactive oxygen species (ROS) formation and gene expression were examined. Results GRP78 levels were higher in blood samples of patients with CAD than in patients without CAD (2584 ng/ml vs. 2061 ng/ml, p = 0.047). In patients presenting with ACS, GRP78 levels were lower than in patients presenting with chronic coronary syndrome. Elevated GRP78 levels were independently associated with lower one-year mortality, even after adjustment for cardiovascular risk factors and demographic parameters (4.5% vs. 9.0%, log-rank p = 0.022; Fig.1A). Increased levels of GRP78 (> median of 1710 ng/ml) were associated with a higher BMI (29.0 kg/m2 ± 7.3 vs. 27.0 kg/m2 ± 6.2, p < 0.0001) while there was no difference regarding age, sex, or cardiovascular risk factors. Mass spectrometry analyses of the HCAEC secretome and ELISA revealed increased extracellular GRP78 secretion after ER-Stress activation. Co-incubation with Brefeldin A, an inhibitor of ER-Golgi-trafficking, confirmed active regulation of GRP78 secretion. GRP78-containing CM improved viability while reducing apoptosis, formation of ROS, ER-Stress-signaling, and inflammation in recipient HCAEC compared to CM lacking GRP78 due to BFA treatment or GRP78-knockdown. Likewise, recombinant GRP78 improved HCAEC viability while reducing expression of markers of inflammation and ER stress (Fig.1B). CRIPTO is a membrane-bound co-receptor for growth factors and was previously linked to GRP78-signalling. siRNA-Knockdown of CRIPTO reduces viability and leads to cell apoptosis, irrespective of GRP78-treatment (relative viability: Scr-siRNA+1µg/ml GRP78 vs. Criptodown+1µg/ml GRP78 1.22 ± 0.23 vs. 1.02 ± 0.13, p<0.01, Fig.1C). Thus, GRP78-mediated effects may be mediated by CRIPTO. Conclusion GRP78 is significantly increased in patients with CAD. Intriguingly, elevated levels are associated with reduced one-year mortality. ER-Stress-induced GRP78 secretion represents a feedback mechanism to ameliorate ER-Stress and inflammation in recipient endothelial cells (Fig.2).

Selective bioorthogonal probe for N-glycan hybrid structures.

Metabolic incorporation of chemically tagged monosaccharides is a facile means of tagging cellular glycoproteins and glycolipids. However, since the monosaccharide precursors are often shared by several pathways, selectivity has been difficult to attain. For example, N-linked glycosylation is a chemically complex and ubiquitous posttranslational modification, with three distinct classes of GlcNAc-containing N-glycan structures: oligomannose, hybrid and complex. Here we describe the synthesis of 1,3-Pr2-6-OTs GlcNAlk (MM-JH-1) as a next-generation metabolic chemical reporter for the selective labeling of hybrid N-glycan structures. We first developed a general strategy for defining the selectivity of labeling with chemically tagged monosaccharides. We then applied this approach to establish that MM-JH-1 is selectively incorporated into hybrid N-glycans. Using this metabolic chemical reporter as a detection tool, we performed imaging and fractionation to define features of the intracellular localization and trafficking of target proteins bearing hybrid N-glycan structures.

Biosynthesis of pH-Responsive Mesoporous Silica Nanoparticles from Cucumber Peels for Targeting 3D Lung Tumor Spheroids.

Lung adenocarcinoma is considered to be one of the primary causes of cancer-related deaths globally. Conventional treatments, such as chemotherapy and radiation therapy taken together, have not significantly lowered mortality rates. Repositioning of authorized anticancer medications supported by nanotechnology has therefore emerged as an effective strategy to close such gaps. In this context, mesoporous silica nanoparticles (MSNs) were biosynthesized from cucumber peels and were loaded with doxorubicin, a common anticancer drug to form doxorubicin-bound mesoporous silica nanoparticles (DMSNs). The study addresses a sustainable method for turning waste materials into MSNs, which can be used to create multifunctional nanosystems. The therapeutic module (DMSNs) was designed specifically to target 2D monolayer cells and 3D tumor spheroids of lung adenocarcinoma cancer. The DMSNs demonstrated notable antiproliferative activity and effective intracellular localization in addition to being biocompatible and innately fluorescent. Subsequent investigations revealed significant antibacterial activity against Staphylococcal infection, which is primarily prevalent in lung cancer patients. Thus, the developed MSNs held promising potential for anticancer drug delivery systems and have antibacterial potential to treat bacterial infections in patients with lung cancer. Furthermore, the cucumber peel-mediated synthesis of MSNs could also aid in the management of food waste and promote the adoption of the waste-to-health paradigm for sustainable solutions.

Partial deletion of the Akap1 gene promotes abnormalities in gut barrier function, gut microbiota composition and cardiac function during cardiovascular aging in mice

Abstract Background Cardiovascular aging is characterized by mitochondrial dysfunction, changes in intestinal permeability and microbiota composition. Mitochondria-targeted A-kinase anchoring proteins (mitoAKAPs) are a group of structurally diverse proteins binding the regulatory subunits of protein kinase A (PKA), and targeting PKA at discrete intracellular locations. We analyzed the effects of global partial genetic deletion of Akap1 on intestinal barrier permeability, microbiota composition, inflammation, and cardiac function during aging in mice. Purpose The proposal of this study was to determine the role of mitoAKAPs in the gut-heart axis during cardiovascular aging. Methods Young (4-6-month) and old (18-24-month) genetically modified Akap1+/+ and Akap1+/− mice of either sex underwent evaluation of cardiac function by transthoracic echocardiography. To evaluate gut barrier integrity, we analyzed expression levels of intestinal junction proteins occludin (Ocln), zonulin (Tjp1) in colonic samples and FITC-dextran permeability in vivo, circulating levels of Tumor Necrosis Factor-alpha (TNF-alpha), Lipopolysaccharide (LPS), Interleukin-1 (IL-1) and Interleukin-10 (IL-10). Fecal microbial composition was evaluated by Illumina Mi-Seq, Gut microbiota differences based on 16S rDNA sequencing at genus and species taxonomic levels were identified using linear discriminant analysis (LDA) combined with effect size (LEfSe) algorithm. Finally, faecal microbiota transplantation (FMT) was performed for five weeks to test whether modification of gut microbiota composition can affect cardiac function. Results A reduction in % left ventricular shortening (FS%) was observed in young and old Akap1+/- mice compared to Akap1+/+ mice. This finding was associated to increased intestinal permeability, as indicated by reduced mRNA levels of Ocln and Tjp1, increased LPS traslocation across intestinal epitelium into blood in 24m Akap1+/- mice compared to Akap1+/+. Through the analysis of the microbial signature, we observed the different bacterial species present in the microbiota of the experimental groups. FMT of bacteria species from old Akap1+/− to young Akap1+/+ mice induced gut abnormalities and cardiac dysfunction, while FMT from young Akap1+/+ donors ameliorated cardiac dysfunction in old Akap1+/+ mice. Conclusions MitoAKAPs play a crucial role in the maintenance of intestinal barrier function, gut microbiota composition and cardiac function during aging. Modulation of the composition of the intestinal microbiota influences intestinal permeability and cardiac function. MitoAKAPs could represent an important diagnostic and therapeutic target for cardiac and intestinal dysfunction.

Multiple functions of the herpesvirus UL14 gene product in viral infection.

Herpesviruses are a family of double-stranded DNA viruses with a tegument structure and a genome composed of a single sequence and terminal repeat (TR) sequences. The herpesvirus UL14 gene encodes the protein UL14 (pUL14), which has various subcellular localizations and plays a vital role in regulating immediate-early (IE) gene transcription and expression, influences the intracellular localization patterns of several proteins belonging to the capsid and the DNA packaging machinery, participates in secondary envelopment, and influences viral particle release. Additionally, pUL14 has roles in maintaining cellular homeostasis and preventing apoptosis. This review discusses how pUL14 engages in the life cycle of herpesviruses and provides new ideas for further research on pUL14's function in viral infection.

Glycopolymeric Nanoparticles Enrich Less Immunogenic Protein Coronas, Reduce Mononuclear Phagocyte Clearance, and Improve Tumor Delivery Compared to PEGylated Nanoparticles.

Nanoparticles (NPs) offer significant promise as drug delivery vehicles; however, their in vivo efficacy is often hindered by the formation of a protein corona (PC), which influences key physiological responses such as blood circulation time, biodistribution, cellular uptake, and intracellular localization. Understanding NP-PC interactions is crucial for optimizing NP design for biomedical applications. Traditional approaches have utilized hydrophilic polymer coatings like polyethylene glycol (PEG) to resist protein adsorption, but glycopolymer-coated nanoparticles have emerged as potential alternatives due to their biocompatibility and ability to reduce the adsorption of highly immunogenic proteins. In this study, we synthesized and characterized glycopolymer-based poly[2-(diisopropylamino)ethyl methacrylate-b-poly(methacrylamidoglucopyranose) (PDPA-b-PMAG) NPs as an alternative to PEGylated NPs. We characterized the polymers using a range of techniques to establish their molecular weight and chemical composition. PMAG and PEG-based NPs showed equivalent physicochemical properties with sizes of ∼100 nm, spherical morphology, and neutral surface charges. We next assessed the magnitude of protein adsorption on both NPs and catalogued the identity of the adsorbed proteins using mass spectrometry-based techniques. The PMAG NPs were found to adsorb fewer proteins in vitro as well as fewer immunogenic proteins such as Immunoglobulins and Complement proteins. Flow cytometry and confocal microscopy were employed to examine cellular uptake in RAW 264.7 macrophages and MDA-MB-231 tumor cells, where PMAG NPs showed higher uptake into tumor cells over macrophages. In vivo studies in BALB/c mice with orthotopic 4T1 breast cancer xenografts showed that PMAG NPs exhibited prolonged circulation times and enhanced tumor accumulation compared to PEGylated NPs. The biodistribution analysis also revealed greater selectivity for tumor tissue over the liver for PMAG NPs. These findings highlight the potential of glycopolymeric NPs to improve tumor targeting and reduce macrophage uptake compared to PEGylated NPs, offering significant advancements in cancer nanomedicine and immunotherapy.

Tryptophanol-derived oxazoloisoindolinone fluorescent probes for cellular localization studies of p53 activators

The protein p53 is a transcription factor with several key roles in cells, including acting as a tumour suppressor. In most human cancers its tumour suppressor function is inactivated, either through inhibition by negative regulators or by mutation in the TP53 gene. Thus, there is a high interest in developing molecules able to activate p53 tumour suppressor activity. Tryptophanol-derived isoindolinones are known to act as wild-type and mutant p53 activators. Specifically, SLMP53-1 is a non-fluorescent wild-type and mutant p53 R280K reactivator, with potent in vivo anti-tumour activity in HCT116 and MDA-MB-231 mice xenograft models. With the aim of studying tryptophanol-derived isoindolinones intracellular localization by fluorescence microscopy, three SLMP53-1 based fluorescent probes were prepared. Here we report the design, synthesis, photophysical characterization, antiproliferative activity and cell localization studies of these fluorescent probes.The previously described structure–activity relationships of the SLMP53-1 scaffold set the basis for the design the fluorescent probes. The probes were prepared by connecting a small fluorophore (dansyl or 7-nitrobenzofurazan) to the indole nitrogen of the tryptophanol-derived oxazoloisoindolinone SLMP53-1 through two different linkers. The antiproliferative activity and cell localization studies of the three fluorescent probes were performed in HCT116 cells. The three probes showed enhanced internalization when compared with their fluorophore-linker intermediates, good photo-stability and high affinity for the endoplasmic reticulum, indicating the potential involvement of endoplasmic reticulum in the mechanism of action of tryptophanol-derived oxazoloisoindolinones.

Analysis of low-density lipoprotein receptor gene mutations in a family with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a common monogenic autosomal dominant disorder, primarily mainly caused by pathogenic mutations in the low-density lipoprotein receptor (LDLR) gene. Through phenotypic-genetic linkage analysis, two LDLR pathogenic mutations were identified in FH families: c.G1027A (p.Gly343Ser) and c.G1879A (p.Ala627Thr). Whole exome sequencing was conducted on the proband with familial hypercholesterolemia to identify the target gene and screen for potential pathogenic mutations. The suspicious responsible mutation sites in 14 family members were analyzed using Sanger sequencing to assess genotype-phenotype correlations. Mutant and wild type plasmids were constructed and transfected into HEK293T cells to evaluate LDLR mRNA and protein expression. In parallel, bioinformatics tools were employed to predict structural and functional changes in the mutant LDLR. Immunofluorescence analysis revealed no significant difference in the intracellular localization of the p.Gly343Ser mutation, whereas protein expression of the p.Ala627Thr mutation was decreased and predominantly localized in the cytoplasm. Western blotting has showed that protein expression levels of the mutant variants were markedly declined in both cell lysates and supernatants. Enzyme linked immunosorbent assay has demonstrated that LDLR protein levels in the supernatant of cell culture medium was not significant different from those of the wild-type group. However, LDLR protein levels in the cell lysate of both the Gly343Ser and Ala627Thr variants groups were significantly lower than those in the wild-type group. Bioinformatic predictions further suggested that these mutations may affect post-translational modifications of the protein, providing additional insight into the mechanisms underlying the observed reduction in protein expression. In this study, we identified two heterozygous pathogenic variants in the LDLR gene, c.G1027A (p.Gly343Ser) and c.G1879A (p.Ala627Thr), in a family with familial hypercholesterolemia. We also conducted preliminary investigations into the mechanisms by which these mutations contribute to disease pathology.

8543 Non-Specific Uptake Of Magnetic Iron Oxide Nanoparticles Into Adrenocortical Carcinoma Cells, Endothelium And Primary Monocytes

Abstract Disclosure: R. Challapalli: None. A. Sorushanova: None. C. Hong: None. N.J. Mullen: None. S. Feely: None. O. Covarrubias-Zambrano: None. J. Covarrubias: None. S. Varghese: None. C. Hantel: None. P. Owens: None. M. O’Halloran: None. P. Prakash: None. S.H. Bossmann: None. M.C. Dennedy: None. Adrenocortical carcinoma (ACC), a rare malignancy with a poor prognosis and a survival rate of up to 24 months, requires varied treatments such as surgery, chemotherapy, or radiation. The high recurrence rate emphasizes the need for innovative therapies. Magnetic iron oxide nanoparticles have emerged as promising candidates for cancer treatment due to their versatility in size and shape and the ability to modify their surface for targeted cellular uptake. In our initial investigation, we assessed the uptake of magnetic iron oxide nanoparticles (IONP) by ACC (H295R, HAC15, and MUC1) cells, HUVECs and primary monocytes, incubated with 0, 5, 10, 20, and 50 µg/ml of IONP for 24 hours. The results demonstrated concentration and time-dependent nanoparticle uptake and its impact on cellular viability. The optimal concentration of IONP was identified as 10 µg/ml, subsequently used to evaluate the rate of uptake and intracellular location. The efficiency of IONP uptake by ACC cells decreased when exposed to primary monocytes and an HUVEC layer, as demonstrated through a Transwell migration system. This study revealed insights into the non-specific uptake of IONP by ACC, primarily attributed to the mechanism of macropinocytosis. These preliminary findings lay the groundwork for exploring potential modifications to enhance specific uptake by ACC cells, particularly for applications like magnet-induced thermal ablation or nanobiocontrast. Presentation: 6/2/2024